development of an icelisa and immunochromatographic assay for methyl-3-quinoxaline-2-carboxylic acid residues in fish

To evaluate residues of olaquindox (ola) and its marker compound methyl-3-quinoxaline-2-carboxylic acid (mqca) in aquatic products in the field, five types of hapten and corresponding antigens with exposure of different antigenic determinants were designed and synthesized. the optimal immunogen-coating combinations were obtained by antiserum detection and heterologous coating screening, successfully achieving a highly specific monoclonal antibody (mab) against mqca. based on the highly specific mab 1a11 showing no cross-reactivity with other analogs, an indirect competitive elisa and an immunochromatographic strip for mqca detection in fish were developed using the mqca derivative mqca-(nh2)4 coupled to klh as the immunogen and mqca-nh2 coupled to ova as the heterologous coating antigen. the ic50 value of the indirect competitive enzyme-linked immunosorbent assay (icelisa) was 3.17 ng/ml, and the linear range was 0.58–17.29 ng/ml for mqca under optimized concentrations of coating antigen and antibody of 0.03 and 0.3 μg/ml, respectively. the recoveries of mqca by the developed icelisa in fish samples ranged from 81.6 to 90.7%. the cutoff value of the strip was 5 ng/ml, and the limit of detection was 0.25 ng/ml under optimal working conditions of 4 μl 0.1 m k2co3 and 50 μl 0.2 mg/ml per 1 ml colloidal gold solution. the developed icelisa and immunochromatographic strip will be very useful tools for the primary screening of mqca in fish.